Label-Free Structural Imaging of Plant Roots and Microbes Using Third-Harmonic Generation Microscopy
Authors:
Na Ji1,2,4* ([email protected], PI), Daisong Pan1, Jose A. Rivera1, Peter Kim1,2, Max Miao3, Tomas Tyml3, Cristina Rodriguez1, Nathalie H. Elisabeth2, John Vogel3, Trent Northen2
Institutions:
1Department of Physics, University of California–Berkeley; 2Lawrence Berkeley National Laboratory; 3DOE Joint Genome
Institute; 4Department of Molecular and Cell Biology, University of California–Berkeley
Abstract
Root biology is pivotal in addressing global challenges, including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to difficulties in imaging root structures in their natural environment. Here, researchers used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution.
THG enabled researchers to observe key plant root features in mature and meristem roots including laminar structures down to the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as resolving subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provide label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF fluorescence signal, the team demonstrated its ability to investigate root-microbe interactions by achieving single- bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.